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Item Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant(Frontiers Media, 2022-03-09) Fischer, Jonathan T.; Söll, Dieter; Tharp, Jeffery M.; Biochemistry and Molecular Biology, School of MedicinePyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of Methanomethylophilus alvus PylRS (MaPylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of MaPylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve MaPylRS to efficiently incorporate N ε-Boc-l-lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRSopt) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRSopt can be used to enhance previously engineered PylRS constructs such as MaPylRSN166S, and PylRSopt is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.Item Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry(American Chemical Society, 2024-06-04) Jiang, Yuming; Rex, Devasahayam Arokia Balaya; Schuster, Dina; Neely, Benjamin A.; Rosano, Germán L.; Volkmar, Norbert; Momenzadeh, Amanda; Peters-Clarke, Trenton M.; Egbert, Susan B.; Kreimer, Simion; Doud, Emma H.; Crook, Oliver M.; Yadav, Amit Kumar; Vanuopadath, Muralidharan; Hegeman, Adrian D.; Mayta, Martín L.; Duboff, Anna G.; Riley, Nicholas M.; Moritz, Robert L.; Meyer, Jesse G.; Biochemistry and Molecular Biology, School of MedicineProteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics.Item Phosphorylation of GCN2 by mTOR confers adaptation to conditions of hyper-mTOR activation under stress(Elsevier, 2024) Darawshi, Odai; Yassin, Olaya; Shmuel, Miri; Wek, Ronald C.; Mahdizadeh, S. Jalil; Eriksson, Leif A.; Hatzoglou, Maria; Tirosh, Boaz; Biochemistry and Molecular Biology, School of MedicineAdaptation to the shortage in free amino acids (AA) is mediated by 2 pathways, the integrated stress response (ISR) and the mechanistic target of rapamycin (mTOR). In response to reduced levels, primarily of leucine or arginine, mTOR in its complex 1 configuration (mTORC1) is suppressed leading to a decrease in translation initiation and elongation. The eIF2α kinase general control nonderepressible 2 (GCN2) is activated by uncharged tRNAs, leading to induction of the ISR in response to a broader range of AA shortage. ISR confers a reduced translation initiation, while promoting the selective synthesis of stress proteins, such as ATF4. To efficiently adapt to AA starvation, the 2 pathways are cross-regulated at multiple levels. Here we identified a new mechanism of ISR/mTORC1 crosstalk that optimizes survival under AA starvation, when mTORC1 is forced to remain active. mTORC1 activation during acute AA shortage, augmented ATF4 expression in a GCN2-dependent manner. Under these conditions, enhanced GCN2 activity was not dependent on tRNA sensing, inferring a different activation mechanism. We identified a labile physical interaction between GCN2 and mTOR that results in a phosphorylation of GCN2 on serine 230 by mTOR, which promotes GCN2 activity. When examined under prolonged AA starvation, GCN2 phosphorylation by mTOR promoted survival. Our data unveils an adaptive mechanism to AA starvation, when mTORC1 evades inhibition.Item RISING STARS: Evidence for established and emerging forms of β-cell death(Bioscientifica, 2024-07-04) Colglazier, Kaitlyn A.; Mukherjee, Noyonika; Contreras, Christopher J.; Templin, Andrew T.; Biochemistry and Molecular Biology, School of Medicineβ-Cell death contributes to β-cell loss and insulin insufficiency in type 1 diabetes (T1D), and this β-cell demise has been attributed to apoptosis and necrosis. Apoptosis has been viewed as the lone form of programmed β-cell death, and evidence indicates that β-cells also undergo necrosis, regarded as an unregulated or accidental form of cell demise. More recently, studies in non-islet cell types have identified and characterized novel forms of cell death that are biochemically and morphologically distinct from apoptosis and necrosis. Several of these mechanisms of cell death have been categorized as forms of regulated necrosis and linked to inflammation and disease pathogenesis. In this review, we revisit discoveries of β-cell death in humans with diabetes and describe studies characterizing β-cell apoptosis and necrosis. We explore literature on mechanisms of regulated necrosis including necroptosis, ferroptosis and pyroptosis, review emerging literature on the significance of these mechanisms in β-cells, and discuss experimental approaches to differentiate between various mechanisms of β-cell death. Our review of the literature leads us to conclude that more detailed experimental characterization of the mechanisms of β-cell death is warranted, along with studies to better understand the impact of various forms of β-cell demise on islet inflammation and β-cell autoimmunity in pathophysiologically relevant models. Such studies will provide insight into the mechanisms of β-cell loss in T1D and may shed light on new therapeutic approaches to protect β-cells in this disease.Item "How Can I Help?" How Safety Professionals and Institutional Leaders Can Promote Laboratory Safety through Diversity, Equity, Inclusion, and Respect(American Chemical Society, 2024) Dunn, Anna L.; Decker, Debbie M.; Hunter, Kirk; Kimble-Hill, Ann; Maclachlan, Jennifer L.; Situma, Catherine; Biochemistry and Molecular Biology, School of MedicineItem Chloroacetamide fragment library screening identifies new scaffolds for covalent inhibition of the TEAD·YAP1 interaction(Royal Society of Chemistry, 2023-08-03) Bum-Erdene, Khuchtumur; Ghozayel, Mona K.; Zhang, Mark J.; Gonzalez-Gutierrez, Giovanni; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of MedicineTranscriptional enhanced associate domain (TEAD) binding to co-activator yes-associated protein (YAP1) leads to a transcription factor of the Hippo pathway. TEADs are regulated by S-palmitoylation of a conserved cysteine located in a deep well-defined hydrophobic pocket outside the TEAD·YAP1 interaction interface. Previously, we reported the discovery of a small molecule based on the structure of flufenamic acid that binds to the palmitate pocket, forms a covalent bond with the conserved cysteine, and inhibits TEAD4 binding to YAP1. Here, we screen a fragment library of chloroacetamide electrophiles to identify new scaffolds that bind to the palmitate pocket of TEADs and disrupt their interaction with YAP1. Time- and concentration-dependent studies with wild-type and mutant TEAD1-4 provided insight into their reaction rates and binding constants and established the compounds as covalent inhibitors of TEAD binding to YAP1. Binding pose hypotheses were generated by covalent docking revealing that the fragments and compounds engage lower, middle, and upper sub-sites of the palmitate pocket. Our fragments and compounds provide new scaffolds and starting points for the design of derivatives with improved inhibition potency of TEAD palmitoylation and binding to YAP1.Item Transthyretin: a review from a structural perspective(Springer, 2001) Hamilton, J. A.; Benson, M. D.; Biochemistry and Molecular Biology, School of MedicineTransthyretin (formerly called prealbumin) plays important physiological roles as a transporter of thyroxine and retinol-binding protein. X-ray structural studies have provided information on the active conformation of the protein and the site of binding of both ligands. Transthyretin is also one of the precursor proteins commonly found in amyloid deposits. Both wild-type and single-amino-acid-substituted variants have been identified in amyloid deposits, the variants being more amyloidogenic. Sequencing of the gene and the resulting production of a transgenic mouse model have resulted in progress toward solving the mechanism of amyloid formation and detecting the variant gene in individuals at risk.Item Surviving and Adapting to Stress: Translational Control and the Integrated Stress Response(Mary Ann Liebert, 2023) Wek, Ronald C.; Anthony, Tracy G.; Staschke, Kirk A.; Biochemistry and Molecular Biology, School of MedicineSignificance: Organisms adapt to changing environments by engaging cellular stress response pathways that serve to restore proteostasis and enhance survival. A primary adaptive mechanism is the integrated stress response (ISR), which features phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2). Four eIF2α kinases respond to different stresses, enabling cells to rapidly control translation to optimize management of resources and reprogram gene expression for stress adaptation. Phosphorylation of eIF2 blocks its guanine nucleotide exchange factor, eIF2B, thus lowering the levels of eIF2 bound to GTP that is required to deliver initiator transfer RNA (tRNA) to ribosomes. While bulk messenger RNA (mRNA) translation can be sharply lowered by heightened phosphorylation of eIF2α, there are other gene transcripts whose translation is unchanged or preferentially translated. Among the preferentially translated genes is ATF4, which directs transcription of adaptive genes in the ISR. Recent Advances and Critical Issues: This review focuses on how eIF2α kinases function as first responders of stress, the mechanisms by which eIF2α phosphorylation and other stress signals regulate the exchange activity of eIF2B, and the processes by which the ISR triggers differential mRNA translation. To illustrate the synergy between stress pathways, we describe the mechanisms and functional significance of communication between the ISR and another key regulator of translation, mammalian/mechanistic target of rapamycin complex 1 (mTORC1), during acute and chronic amino acid insufficiency. Finally, we discuss the pathological conditions that stem from aberrant regulation of the ISR, as well as therapeutic strategies targeting the ISR to alleviate disease. Future Directions: Important topics for future ISR research are strategies for modulating this stress pathway in disease conditions and drug development, molecular processes for differential translation and the coordinate regulation of GCN2 and other stress pathways during physiological and pathological conditions.Item Exploring Unconventional SAM Analogues To Build Cell-Potent Bisubstrate Inhibitors for Nicotinamide N-Methyltransferase(Wiley, 2022) Iyamu, Iredia D.; Vilseck, Jonah Z.; Yadav, Ravi; Noinaj, Nicholas; Huang, Rong; Biochemistry and Molecular Biology, School of MedicineNicotinamide N-methyltransferase (NNMT) methylates nicotinamide and has been associated with various diseases. Herein, we report the first cell-potent NNMT bisubstrate inhibitor II399, demonstrating a Ki of 5.9 nM in a biochemical assay and a cellular IC50 value of 1.9 μM. The inhibition mechanism and cocrystal structure confirmed II399 engages both the substrate and cofactor binding pockets. Computational modeling and binding data reveal a balancing act between enthalpic and entropic components that lead to II399′s low nM binding affinity. Notably, II399 is 1 000-fold more selective for NNMT than closely related methyltransferases. We expect that II399 would serve as a valuable probe to elucidate NNMT biology. Furthermore, this strategy provides the first case of introducing unconventional SAM mimics, which can be adopted to develop cell-potent inhibitors for other SAM-dependent methyltransferases.Item Immunoglobulin G (IgG) specific responses to recombinant Qβ displayed MSP3 and UB05 in plasma of asymptomatic Plasmodium falciparum-infected children living in two different agro-ecological settings of Cameroon(PAMJ, 2024-04-09) Ngu, Loveline; Fotso, Herve Ouambo; Nyebe, Inès; Tchadji, Jules Colince; Ambada, Georgia; Ndah, Akeleke; Atechi, Bloomfield; Lissom, Abel; Atabonkeng, Philémon Etienne; Chukwuma, George; Efezeuh, Vitalis; Gyu, Park Chae; Esimone, Charles; Assob Nguedia, Jules Clement; Akum, Eric Achidi; Okeke, Malachy; Kehdingha Titanji, Vincent Pryde; Mbacham, Wilfred; Bopda-Waffo, Alain; Wapimewah, Godwin Nchinda; Biochemistry and Molecular Biology, School of MedicineIntroduction: in areas with intense perennial malaria transmission, limited data is available on the impact of environmental conditions especially rainfall on naturally acquired immunity against promising malaria vaccine candidates. For this reason, we have compared IgG antibody responses specific to Plasmodium spp. derived MSP3 and UB05 vaccine candidates, in plasma of children living in two areas of Cameroon differing in rainfall conditions. Methods: data about children less than 5 years old was collected during the years 2017 and 2018. Next malaria asymptomatic P. falciparum (Pf) infected children were selected following malaria test confirmation. MSP3 and UB05 specific IgG antibody responses were measured in participant´s plasma using enzyme-linked immunosorbent assay (ELISA). Results: interestingly, IgG antibody responses specific to UB05 were significantly higher (p<0.0001) in Pf-negative children when compared to their asymptomatic Pf-infected counterparts living in monomodal rainfall areas. In contrast, a significantly higher (p<0.0001) IgG response to MSP3 was observed instead in asymptomatic Pf-infected children in the same population. In addition, IgG responses specific to UB05 remained significantly higher in bimodal when compared to monomodal rainfall areas irrespective of children´s Pf infection status (p<0.0055 for Pf-positive and p<0.0001 for negative children). On the contrary, IgG antibody responses specific to MSP3 were significantly higher in bimodal relative to monomodal rainfall areas (P<0.0001) just for Pf-negative children. Conclusion: thus IgG antibody responses specific to UBO5 are a better correlate of naturally acquired immunity against malaria in Pf-negative Cameroonian children especially in monomodal rainfall areas.